A variety of techniques and devices are commercially available for the detection and measurement of substances present in fluid or other translucent samples by determining the light transmissivity of the sample. These can be broadly categorized into devices which measure the optical properties of one sample or a small number of samples essentially simultaneously and those which measure a large number of samples essentially simultaneously.
Photometric devices that simultaneously perform individual assays on a plurality of liquid or other translucent samples use a multi-assay plate which contains an array of vessels, such as eight rows by twelve columns (8.times.12), four rows by six columns (4.times.6), two rows by three columns (2.times.3), five rows by eight columns (5.times.8). Typically, the vessels of the multi-assay plate are spaced at nine (9) millimeter centers, have a volume of approximately four hundred (400) milliliters, and have a height of approximately one (1) centimeter. The multi-assay plate is made of material, such as polystyrene or polyethylene, that is optically transparent at the wavelengths of interest.
The optical density of the samples is measured by determining the amount of light attenuation. Light passing through the translucent samples, contained in the multi-assay plate vessels, is compared to a reference by conventional photodetectors.
A widespread use of multi-assay plates is in the enzyme-linked immunosorbent assay (ELISA) technique which is used for detection and quantitation of an extensive range of substances and biological cells in academic research and biotechnology as well as for clinical testing. In such assays, molecules of a marker enzyme (such as alkaline phosphatase) are deposited on the bottom and part way up the sides of each of the vessels of a multi-assay plate; each vessel having been assigned to interact previously, directly or indirectly, with a sample containing an analyte of interest. The number of marker enzyme molecules bound to each vessel of the plate is a function of the concentration of analyte in the sample of interest. Determination of the activity of the bound enzyme, therefore, permits detection or quantitation of the analyte.
For determination of fluid-phase enzyme activity, current techniques for both research and clinical applications employ kinetic analysis which involves measurement of the initial rate of enzyme-catalyzed, chromogenic reactions in the presence of excess of the enzyme substrate; a procedure which has several well known advantages over the alternative "end-point" analysis method of allowing the enzyme to react with a chromogenic substrate for a fixed period of time and then making a single optical density measurement after quenching the enzymes. In kinetic analysis, multiple readings are made within the interval (typically linear) reaction period and the intervals between readings are necessarily short (typically less than 30 seconds). By using kinetic analysis, the introduction of errors caused by (a) differences in initial optical density and/or (b) loss of independence from substrate concentration, is substantially avoided. Examples include the use of NADH and NADPH, as described, for example, in Lehninger, "Biochemistry, the Molecular Basis of Cell Structure and Function," Worth Publishers Inc., New York, 1970, and the teachings thereof are expressly incorporated herein by reference. Photometers capable of measuring the absorbance of ultraviolet light by NADH or NADPH at about 340 nanometers wavelength are particularly useful in performing such assays.
Currently available automated optical density measurement instruments for multi-assay plates regulate the temperature of the plate with radiant heating, i.e., heating a metal surface and radiating this heat onto the multi-assay plate. Alternatively, the multi-assay plates are heated by air convection, i.e., heating air and forcing it past the plate. A limitation of the existing air convection heating technology is that the plate takes a long time to warm up and reach equilibrium, adding to the time required to take measurements. Another limitation of existing technology is that the outer vessels, having more exposed surface area, warm up more quickly than the inner vessels and reach a different equilibrium temperature. The reaction rate and the rate of change of optical absorption for some specimens depend upon the temperature of the specimen. In a kinetic test of such specimens, a temperature difference between vessels leads to an erroneous result. Another limitation of existing technology is that moisture can condense on the optical components causing loss of focus and attenuation due to scattering of the light signal. Solvents contained in the moisture can cause corrosion on the surfaces of the optical, mechanical, and electrical components leading to further attenuation of the light signal and eventually to failure of the instrument.
Existing photometric devices capable of measuring multi-assay plates have been limited in the range and selectivity of the light spectrum that is provided for measurement and analysis. This limitation arises because the light source needs to be distributed to a plurality of samples, typically requiring multiple distribution elements and a vertical illumination of the samples. Proteins and Deoxyribonucleic Acids (DNAs) as well as many chromogenic substrates absorb at wavelengths shorter than 340 nanometers. Currently available photometric instruments that can operate at wavelengths less than approximately 340 nanometers are limited to assays on one or a small numbers of samples, thereby making large scale kinetic analysis assay applications impractical due to the extended sampling times. Additionally, currently available photometric instruments are limited to analysis at a single or small number of excitation wavelengths, thereby making overall full spectrum analysis impractical.
Another limitation associated with conventional photometric devices, when used for assaying chromogenic reactions kinetically, is that the conventional devices are subject to errors arising from erratic redistribution of the colored product as a result of phase separation and/or uncontrolled bulk movement of the aqueous phase of the sample during kinetic analysis. More specifically, in the case of ELISA protocols, where the enzyme is bound to the plastic surface of the multi-assay plate vessels (on the bottom and part way up the sides), the bound enzyme interacts with an unstirred aqueous phase layer which causes localized phase separation of the colored product of the enzyme reaction due to its high local concentration. This separation introduces an unquantifiable error and a degree of non-linearity into such kinetic measurements. Even in cases where the colored product remains in true solution, erratic bulk movement of the aqueous phase leads to uneven redistribution of the concentrated product and hence to an unquantifiable error.
Conventional multi-assay plate photometric measuring instruments are further limited in their utility by their use of interference filters in selecting a precise wavelength of test light. Fixed interference filters are constructed to provide a single predetermined test wavelength that cannot be easily changed. The user must change filters to change test wavelengths. Even with a filter wheel, measurements at more than a few wavelengths and spectrum measurements are impractical. Continuously variable interference filters are difficult to manufacture in a precisely reproducible way and photometric instruments with such filters are difficult to calibrate. Below 340 nanometers variable filters are difficult to manufacture so as to have adequate ultraviolet light transmission.